Polyhydroxyalkanoates (hereinafter referred to briefly as “PHA” s) are thermoplastic polyesters which are synthesized and stored as an energy storage substance in cells of a variety of microorganisms. The PHAs, which are produced by microorganisms using natural organic acids or oils as carbon sources, are completely biodegraded by a microorganism in soil or water to be taken up in the carbon cycle of the natural world. Therefore, PHAs can be said to be an environment-conscious plastic material which hardly causes adverse effects for ecological system. In these years, a synthetic plastic came into a serious social problem in view of environment pollution, waste disposal and oil resource, thus the PHA has attracted attention as an eco-friendly green plastic and its practical applications are longed for. Also in the field of medical treatment, it is considered possible to use PHAs as implant materials which do not require recovery, or vehicles for drug. Thus, practical applications thereof have been expected.
Since PHAs synthesized by a microorganism are stored in cells usually in the form of granules, it is required a procedure for separating PHAs from microbial cells to utilize them as plastics. The known technology for separation and purification of PHAs from microbial cells can be roughly classified into technologies which comprise extracting a PHA from microbial cells with organic solvents capable of solving a PHA and technologies which comprise removing cell components other than PHAs by cell disruption or solubilization.
In earlier researches, many technologies for separating and purifying PHAs by extraction using organic solvents were reported (see Japanese Kokai Publication Sho-55-118394, Japanese Kokai Publication Sho-57-65193, Japanese Kokai Publication Sho-63-198991, Japanese Kokai Publication Hei-02-69187 and Japanese Kokai Publication Hei-07-79788). In these reports, halogen compounds such as chloroform were used as organic solvents having the highest solubility of PHAs, but when a PHA was dissolved in such a solvent, viscosity of solution became very high and handling of the solution became difficult. Therefore, for extracting a PHA, it was needed to set the polymer concentration in a range as extremely low as about 2 to 3%, thus significantly large amount of solvent was required. In addition, for crystallizing a PHA from a solvent layer in a high yield, a large amount as 4 to 5 times as the above solvent of poor solvents for a PHA, such as methanol and hexane, were separately required. Accordingly, for production on an industrial scale, large-scale equipment is required. Moreover, a PHA cannot be produced in a low cost since these technologies require huge amount of solvent, and therefore it takes much cost for solvent recovery and cost due to solvent loss. Due to such reasons as mentioned above, these methods have not been put into practice.
On the other hand, various technologies have been reported which comprise solubilizing and removing cell components other than PHAs by chemical treatments or physical disruption treatments to recover PHAs in the form of granules.
As a method for chemically treating a microbial cell (hereinafter, sometimes referred to as “cell”), J. Gen. Microbiology, 1958 vol. 19, p. 198-209 discloses a technology which comprises treating a suspension of a microbial cell with sodium hypochlorite and solubilizing cell components other than a PHA to recover the PHA. In this technology, marked degradation of a PHA is caused in solubilizing the cell components other than the PHA, and processing ways into products are limited. Moreover, sensible smell of chlorine is left behind in PHAs, which is undesirable for a polymer product. Thus, this technology is not considered to be suitable for practical use. Japanese Kokoku Publication Hei-04-61638 discloses a recovering process which comprises heat treatment in combination with use of an enzyme and/or a surfactant. In this process, heating a suspension to 100° C. or above beforehand is required to decompose nucleic acids, since the suspension becomes highly viscous by free nucleic acids when cells are dissolved by an enzyme treatment. However, the molecular weight of a PHA decreases markedly by heating to 100° C. or above, and an application to products will become impossible. Moreover, despite this technology is very complicated and requires many processes, purity of an obtained PHA is as much as about 88% in general, and 97% even at the maximum. Additionally, a technology which comprises treating a PHA-containing microbial cell with a surfactant, decomposing a nucleic acid released from the cell with hydrogen peroxide at 80° C. for 3 hours, and separating a PHA with a purity of 99% (see Japanese Kohyo Publication Hei-08-502415), and a technology which comprises heating a suspension of a PHA-containing microorganism to 50° C. or higher under a strongly acidic condition of below pH 2, and separating a PHA (see Japanese Kokai Publication Hei-11-266891) have been proposed. Under these heating conditions, the molecular weight of the PHA decreases remarkably, therefore even if its purity is improved, applications to products are still impossible.
On the other hand, as a method applying physical disruption treatments, a technology have been reported, which comprises carrying out an alkali addition with a high-pressure disruption or a combination of a high-pressure disruption. Although “Bioseparation”, 1991, vol. 2, p. 95-105 does not describe purity or yield of a polymer, cell components remain in a poly-3-hydroxybutyrate (PHB) fraction and the purity of PHB is presumably not high since high-pressure disruption is carried out under a condition where pH is returned to neutral after adding alkali to a cell suspension containing PHB. Japanese Kokai Publication Hei-07-31487 discloses a technology which comprises heating to 80° C. after an alkali addition to a cell suspension containing a PHA, stirring the mixture for 1 hour and recovering a polymer by centrifugation; Japanese Kokai Publication Hei-07-31488a discloses a technology which comprises carrying out high-pressure disruption at 70° C.; and a technology considered to develop the method described in the above “Bioseparation”, 1991, vol. 2, p. 95-105, that is a technology which comprises carrying out high-pressure disruption at 70° C. or higher after the alkali addition in Japanese Kokai Publication Hei-07-31489, respectively. By these technologies, since the processes are carried out in high temperature conditions, there is a tendency toward remarkable decrease in a molecular weight of a PHA in some conditions. Moreover, purity is also as low as about 66 to 85%, thus these technologies may not be applied to actual industrial processes.
As mentioned above, we can find it very difficult to recover a PHA from a cultured cell without decreasing the molecular weight but with high purity and a high yield in a low-cost on an industrial production.
By the way, when a PHA is obtained by a technology comprising solubilizing to remove cell components other than a PHA by a chemical or physical treatment and recovering the PHA in the form of granules, the obtained PHA usually occurred as a form of fine particles having diameters of several microns. It is more difficult to separate such fine particles from a liquid medium as compared with the case of particles having a larger diameter. Moreover, these fine particles are considered to have problems such as a risk to cause dust explosion and/or accumulation in lungs when aspirated, thus care should be taken for handling.
In order to avoid these problems, there have been attempts to enlarge the particle diameter by agglomerating a PHA. For example, an agglomeration method by heating or an alkaline metal salt has been developed. As a method of agglomeration by heating, a technology is disclosed, which comprises agglomerating PHB by heating a PHB-containing suspension to the vicinity of the melting point of PHB (180° C.) (see Bailey, Neil A.; George, Neil; Niranjan, K.; Varley, Julie. Biochemical Engineering group, University Reading, “I Chem E Res. Event, Eur. Conf. Young Res. Chem. Eng.” (Britain), 2nd edition, Institution of Chemical Engineers, 1996, vol. 1, p. 196-198). Moreover, Japanese Kohyo Publication Hei-07-509131 discloses a technology to enlarge a particle diameter of a copolymer comprised of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) (hereinafter such copolymer is referred to as “PHBV”), which comprises injecting steam with an appropriate temperature and pressure directly into an aqueous suspension of PHBV, and heating -and stirring the suspension at 120 to 160° C. However, they are not practical since these technologies require heating at a high temperature, the molecular weight of PHA decreases remarkably, and further special equipment with a pressure resistance is required. Alternatively, as a method for agglomerating a PHA by adding an alkaline metal salt, a technology for agglomeration using a divalent cation (see J. Biotechnol., 1998, vol. 65 (2, 3), p. 173-182, and Japanese Kohyo Publication Hei-05-0507410) has been disclosed. However, these technologies are not preferable in, for example, that polymer agglomeration strength is not always high, that a metal salt is contaminated into a polymer, and the like ploblems. Alternatively, a technology which comprises agglomerating PHB by blowing ultrafine bubbles into a PHB suspension to raise a flock to the surface (see Spec. Publ.-R. Soc. Chem., 1994, vol. 158 (Separations for Biotechnology 3), p. 113-119) has also been reported. However, the agglomerate obtained by this technology has a diameter of 2 to 45 μm, and cannot be said as a sufficient size.
Thus, any methods for controlling a molecular-weight decrease of a PHA and carrying out agglomeration effectively have not been known in the state of the art.
As described above, there lies a large obstacle for practical applications in studies of PHAs, which are one species of biodegradable polymers derived from microorganisms, since any processes with low costs and suitable for industrial productions have not been established respectively in recovering PHAs from microbial cells, and further in agglomerating PHA particles carried out according to need.